Opiate Antagonists for Chronic Pain: A Review on the Benefits of Low-Dose Naltrexone in Arthritis versus Non-Arthritic Diseases.

Chronic pain conditions create major financial and emotional burdens that can be devastating for individuals and society. One primary source of pain is arthritis, a common inflammatory disease of the joints that causes persistent pain in affected people. The main objective of pharmacological treatments for either rheumatoid arthritis (RA) or osteoarthritis (OA) is to reduce pain. Non-steroidal anti-inflammatory drugs, opioids, and opioid antagonists have each been considered in the management of chronic pain in arthritis patients. Naltrexone is an oral-activated opioid antagonist with biphasic dose-dependent pharmacodynamic effects. The molecule acts as a competitive inhibitor of opioid receptors at high doses. However, naltrexone at low doses has been shown to have hormetic effects and provides relief for chronic pain conditions such as fibromyalgia, multiple sclerosis (MS), and inflammatory bowel disorders. Current knowledge of naltrexone suggests that low-dose treatments may be effective in the treatment of pain perception in chronic inflammatory conditions observed in patients with either RA or OA. In this review, we evaluated the therapeutic benefits of low-dose naltrexone (LDN) on arthritis-related pain conditions.

Effect of LOXL2 on metastasis through remodeling of the cell surface matrix in non-small cell lung cancer cells.

Lung cancer is the prominent cause of cancer-associated death primarily because of distant metastatic disease. The metastatic potential of non-small cell lung cancer (NSCLC) is associated with tumor cell aggregation. However, the systemic mechanotransduction mechanism by which tumor cells dynamically aggregate and disseminate is poorly understood, especially in NSCLC. In this study, we examine whether the cell surface matrix plays an important role in metastasis. We used poly-2-hydroxyethyl methacrylate-based 3D spheroid formation methods to mimic in vivo metastatic lesions. Supra-structural analysis of human NSCLC A549 cells stained with ruthenium red for transmission electron microscopy (TEM) showed that glycocalyx surrounding the cell surface in 2D culture decreases in 3D culture. Comprehensive gene expression analysis revealed that the genes associated with cell adhesion were distinctly enriched in A549 cell spheroids. Of these, downregulation of the tumor metastatic microenvironment facilitator LOXL2, a copper-dependent enzyme catalyzing posttranslational oxidative deamination of peptidyl lysine, was of special interest. Knockdown of LOXL2 thickened the cell surface matrix in 2D culture and impaired compact aggregate formation in 3D culture. Moreover, A549 cell spheroids with endogenous overexpression of LOXL2 increased their dissemination on basement extracellular matrix Matrigel. Overall, these data imply that cell detachment-downregulated LOXL2 contributes to cell surface matrix remodeling, leading to collective dissemination of free-floating aggregates.

AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions.

Tumor cell aggregation is critical for cell survival following the loss of extracellular matrix attachment and dissemination. However, the underlying mechanotransduction of clustering solitary tumor cells is poorly understood, especially in non-small cell lung cancers (NSCLC). Here, we examined whether cell surface protrusions played an important role in facilitating the physical contact between floating cells detached from a substrate. We employed poly-2-hydroxyethyl methacrylate-based 3D culture methods to mimic in vivo tumor cell cluster formation. The suprastructural analysis of human NSCLC A549 cell spheroids showed that finger-like protrusions clung together via the actin cytoskeleton. Time-lapse holotomography demonstrated that the finger-like protrusions of free-floating cells in 3D culture displayed exploratory coalescence. Global gene expression analysis demonstrated that the genes in the organic hydroxyl transport were particularly enriched in the A549 cell spheroids. Particularly, the knockdown of the water channel aquaporin 3 gene (AQP3) impaired multicellular aggregate formation in 3D culture through the rearrangement of the actomyosin cytoskeleton. Moreover, the cells with reduced levels of AQP3 decreased their transmigration. Overall, these data indicate that cell detachment-upregulated AQP3 contributes to cell surface protrusions through actomyosin cytoskeleton remodeling, causing the aggressive aggregation of free-floating cells dependent on the property of the substratum and collective metastasis.

SOX2, a stemness gene, induces progression of NSCLC A549 cells toward anchorage-independent growth and chemoresistance to vinblastine.

BACKGROUND: Non-small cell lung cancer (NSCLC) is difficult to treat successfully. This intractability is mainly due to the cancer progressing through invasion, metastasis, chemotherapeutic resistance and relapse. Stemness has been linked to the various steps of cancer progression in a variety of tumors, yet little is known regarding its role in NSCLC. PURPOSE: In this study, we sought to determine the role of SOX2, a master regulator of pluripotency, in the growth of extracellular matrix (ECM)-detached cells during cancer progression. METHODS: We established a three-dimensional (3D) Poly-2-hydroxyethyl methacrylate (poly-HEMA) culture of lung adenocarcinoma (LUAD) A549 cells as an ECM-detached cell growth model and examined the role of stemness genes using siRNA and small molecule inhibitor in comparison to standard two dimensional (2D) culture. RESULTS: In poly-HEMA culture, A549 cells formed substratum-detached spheroids with characteristics of intermediate epithelial to mesenchymal transition (EMT) and exhibited greater expression of SOX2 than did control 2D cells. Knockdown of SOX2 markedly suppressed the growth of A549 cell aggregates in poly-HEMA culture conditions and furthermore increased their sensitivity to the anticancer drug vinblastine with concomitant downregulation of the activity of the anti-apoptotic AKT kinase. Interestingly, a small molecule, RepSox, which replaces SOX2, stimulated A549 cell growth in poly-HEMA 3D culture condition. CONCLUSION: Our findings strongly indicate that SOX2 contributes to anchorage-independent growth and chemoresistance via its downstream signaling mediator AKT kinase during the disease progression of NSCLC. SOX2 may therefore be an invaluable therapeutic target of NSCLC.

Crosstalk with cancer-associated fibroblasts induces resistance of non-small cell lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibition.

Although lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly sensitive to selective EGFR tyrosine kinase inhibitors (TKIs), these tumors invariably develop acquired drug resistance. Host stromal cells have been found to have a considerable effect on the sensitivity of cancer cells to EGFR TKIs. Little is known, however, about the signaling mechanisms through which stromal cells contribute to the response to EGFR TKI in non-small cell lung cancer. This work examined the role of hedgehog signaling in cancer-associated fibroblast (CAF)-mediated resistance of lung cancer cells to the EGFR TKI erlotinib. PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR TKI erlotinib when cocultured in vitro with CAFs. Polymerase chain reaction and immunocytochemical assays showed that CAFs induced epithelial to mesenchymal transition phenotype in PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition marker proteins including vimentin. Importantly, CAFs induce upregulation of the 7-transmembrane protein smoothened, the central signal transducer of hedgehog, suggesting that the hedgehog signaling pathway is active in CAF-mediated drug resistance. Indeed, downregulation of smoothened activity with the smoothened antagonist cyclopamine induces remodeling of the actin cytoskeleton independently of Gli-mediated transcriptional activity in PC9 cells. These findings indicate that crosstalk with CAFs plays a critical role in resistance of lung cancer to EGFR TKIs through induction of the epithelial to mesenchymal transition and may be an ideal therapeutic target in lung cancer.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation.

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27(Kip1) protein level specifically increased after KIF14 knockdown. The increase in p27(Kip1) was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27(Kip1) accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27(Kip1) for degradation by the 26S proteasome, leading to accumulation of p27(Kip1). The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Establishment and characterization of a lung cancer cell line, SMC-L001, from a lung adenocarcinoma.

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.

Tumor-stromal interactions with direct cell contacts enhance motility of non-small cell lung cancer cells through the hedgehog signaling pathway.

The metastatic potential of non-small cell lung cancer (NSCLC) has been shown to be associated with interactions with the tumor microenvironment, which primarily comprises of cancer-associated fibroblasts (CAFs). Heterotypic cell-cell interactions occur via released signaling molecules and direct physical contact. To investigate the differential contribution of direct cell-cell contact and paracrine signaling factors to NSCLC metastasis, we performed two types of co-cultures: direct co-cultures of the NSCLC cell line H358 with primary cultures of CAFs from patients with resected NSCLC; and indirect co-cultures across a separable membrane. We showed that CAFs more potently induce epithelial-to-mesenchymal transition (EMT) in NSCLC H358 cells through direct contacts than through indirect interactions, as indicated by an elongated and disseminated appearance. Immunocytochemical experiments show that EMT accompanies the expression of mesenchymal cytoskeletal proteins, including vimentin. However, H358 cells proliferate more slowly in direct co-culture than in indirect co-culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that H358 cells in direct contact with CAFs up-regulate the expression of the pan-mesenchymal markers α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), transforming growth factor-ß (TGFß) signaling effector SMAD family number-3 (SMAD3), and hedgehog signaling effector GLI family zinc finger-1 (GLI1), compared with the indirect co-culture system. Furthermore, we found that the direct GLI1 transcription targets snail family zinc finger-1 (SNAI1) and SNAI2 are up-regulated, suggesting that the hedgehog signaling pathway is active in direct co-culture. A scratch wound assay showed that direct contact co-culture increases the motility of H358 cells. In conclusion, these findings provide evidence that paracrine factors and direct physical contact between NSCLC cells and CAFs might control the metastatic potential of NSCLC through the hedgehog signaling pathway.

Human lung cancer-associated fibroblasts enhance motility of non-small cell lung cancer cells in co-culture.

The metastatic potential of non-small cell lung cancer (NSCLC) cells has been shown to be associated with the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, regulating tumor cell function by secreting growth factors, chemokines, and extracellular matrix (ECM). In this study, we examined the role of CAFs in the tumor progression of NSCLC. Firstly, we established primary cultures of CAFs and matched normal fibroblasts (NFs) from patients with resected NSCLC. CAFs exhibited greater expression of the pan-mesenchymal marker α-smooth muscle actin (α-SMA) than did NFs, although they displayed similar morphology. Furthermore, we employed a direct co-culture assay with human NSCLC A549 and H358 cells, and found that CAFs were more potent in inducing the epithelial-to-mesenchymal transition (EMT) phenotype than NFs, as indicated by an elongated and disseminated appearance. CAF-induced EMT led to an increase in motility and a decrease in proliferation of NSCLC cells through SMAD family number-3 (SMAD3)-dependent up-regulation of the growth inhibitory gene p21(CIP1) [cyclin-dependent kinase inhibitor-1A (CDKN1A)] and α-SMA. Taken together, these findings provide evidence that lung CAFs have tumor-promoting capacity distinct from NFs and might play a significant role in the metastatic potential of NSCLC.

Simultaneous copy number gains of NUPR1 and ERBB2 predicting poor prognosis in early-stage breast cancer.

BACKGROUND: The full extent of chromosomal alterations and their biological implications in early breast carcinogenesis has not been well examined. In this study, we aimed to identify chromosomal alterations associated with poor prognosis in early-stage breast cancers (EBC). METHODS: A total of 145 EBCs (stage I and II) were examined in this study. We analyzed copy number alterations in a discovery set of 48 EBCs using oligoarray-comparative genomic hybridization. In addition, the recurrently altered regions (RARs) associated with poor prognosis were validated using an independent set of 97 EBCs. RESULTS: A total of 23 RARs were defined in the discovery set. Six were commonly detected in both stage I and II groups (> 50%), suggesting their connection with early breast tumorigenesis. There were gains on 1q21.2-q21.3, 8q24.13, 8q24.13-21, 8q24.3, and 8q24.3 and a loss on 8p23.1-p22. Among the 23 RARs, copy number gains on 16p11.2 (NUPR1) and 17q12 (ERBB2) showed a significant association with poor survival (P = 0.0186 and P = 0.0186, respectively). The patients simultaneously positive for both gains had a significantly worse prognosis (P = 0.0001). In the independent replication, the patients who were double-positive for NUPR1-ERBB2 gains also had a significantly poorer prognosis on multivariate analysis (HR = 7.31, 95% CI 2.65-20.15, P = 0.0001). CONCLUSIONS: The simultaneous gain of NUPR1 and ERBB2 can be a significant predictor of poor prognosis in EBC. Our study will help to elucidate the molecular mechanisms underlying early-stage breast cancer tumorigenesis. This study also highlights the potential for using combinations of copy number alterations as prognosis predictors for EBC.

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element.

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.

FOXO1 inhibits Runx2 transcriptional activity and prostate cancer cell migration and invasion.

Prostate cancer patients with regional lymph node involvement at radical prostatectomy often experience disease progression to other organs, with the bone as the predominant site. The transcription factor Runx2 plays an important role in bone formation and prostate cancer cell migration, invasion, and metastasis. Here we showed that the forkhead box O (FOXO1) protein, a key downstream effector of the tumor suppressor PTEN, inhibits the transcriptional activity of Runx2 in prostate cancer cells. This inhibition was enhanced by PTEN but diminished by active Akt. FOXO1 bound to Runx2 in vitro and in vivo and suppressed Runx2's activity independent of its transcriptional function. FOXO1 inhibited Runx2-promoted migration of prostate cancer cells, whereas silencing of endogenous FOXO1 enhanced prostate cancer cell migration in a Runx2-dependent manner. Forced expression of FOXO1 also inhibited Runx2-promoted prostate cancer cell invasion. Finally, we found that expression of PTEN and the level of FOXO1 in the nucleus is inversely correlated with expression of Runx2 in a cohort of prostate cancer specimens from patients with lymph node and bone metastasis. These data reveal FOXO1 as a critical negative regulator of Runx2 in prostate cancer cells. Inactivation of FOXO1 due to frequent loss of PTEN in prostate cancer cells may leave the oncogenic activities of Runx2 unchecked, thereby driving promiscuous expression of Runx2 target genes involved in cell migration and invasion and favoring prostate cancer progression.

c-MYC suppresses BIN1 to release poly(ADP-ribose) polymerase 1: a mechanism by which cancer cells acquire cisplatin resistance.

Cancer cells acquire resistance to DNA-damaging therapeutic agents, such as cisplatin, but the genetic mechanisms through which this occurs remain unclear. We show that the c-MYC oncoprotein increases cisplatin resistance by decreasing production of the c-MYC inhibitor BIN1 (bridging integrator 1). The sensitivity of cancer cells to cisplatin depended on BIN1 abundance, regardless of the p53 gene status. BIN1 bound to the automodification domain of and suppressed the catalytic activity of poly(ADP-ribose) polymerase 1 (PARP1, EC 2.4.2.30), an enzyme essential for DNA repair, thereby reducing the stability of the genome. The inhibition of PARP1 activity was sufficient for BIN1 to suppress c-MYC-mediated transactivation, the G(2)-M transition, and cisplatin resistance. Conversely, overexpressed c-MYC repressed BIN1 expression by blocking its activation by the MYC-interacting zinc finger transcription factor 1 (MIZ1) and thereby released PARP1 activity. Thus, a c-MYC-mediated positive feedback loop may contribute to cancer cell resistance to cisplatin.

Differential gene expression activity among species-specific polypyrimidine/polypurine motifs in mu opioid receptor gene promoters.

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. An appropriate understanding of MOR gene expression across species is critical for understanding its analgesic functions in humans. Here, we undertake a cross-species analysis of the polymorphic polypyrimidine/polypurine (PPy/u) motif, a key enhancer of MOR gene expression. The mouse PPy/u motif is highly homologous to those of rat (67%) and human (83%), but drives reporter gene expression tenfold and fivefold more effectively than those of rat and human, respectively. Circular dichroism profiles of PPy/u oligonucleotides from different species showed that they are primarily different in structure. Conformational studies of reporter plasmids using confocal Raman spectra, S1 nuclease and restriction enzymes demonstrated that the structural difference is the result of changes in the phosphodiester backbone. Furthermore, these conformational disparities produce differences in torsional stress, as shown by topoisomerase II relaxation and activation of different levels of gene expression under hypertonic conditions. This study demonstrates that homologous PPy/u motifs adopt unique species-specific conformations with different mechanisms and activities for gene expression. We further discuss how structural aspects of transcription regulatory elements, rather than the sequence itself, are significant when studying functional gene expression regulatory elements.

S-Adenosylmethionine decarboxylase partially regulates cell growth of HL-60 cells by controlling the intracellular ROS level: Early senescence and sensitization to gamma-radiation.

S-Adenosylmethionine decarboxylase (SAMDC) is a key enzyme for the biosynthesis of spermidine. SAMDC-suppressed HL-60 cells overproduced intracellular reactive oxygen species (ROS), which led to cell growth defect and partial cell death. ROS overproduction was caused by a decrease of the total glutathione (GSH) and the ratio of reduced to oxidized GSH, and by an increase of the intracellular iron uptake. When analyzed by real-time polymerase chain reaction, the transcripts of the genes involved in the GSH synthesis (gamma-glutamyl cysteine synthetase, GSH synthetase), as well as the gene of the GSH-reducing enzyme (NADP+-dependent isocitrate dehydrogenase), were decreased dramatically in these cells. DNA-repairing genes (ATM, PARP, RAD51 and MSH2) also were not activated transcriptionally. In these situations, excessive ROS induced severe DNA damage, which could not be repaired, and ultimately led the cells to a spontaneous cell death or an early senescence state. For such cells, gamma-radiation and cisplatin, which are direct DNA-damaging agents, were very effective for promoting cell death.

Decreased tumorigenicity of c-Myc-transformed fibroblasts expressing active USF2.

USF is a small family of basic helix-loop-helix leucine zipper (bHLH-zip) transcription factors with DNA binding specificities similar to that of the c-Myc oncoprotein. Evidence for a role of USF in growth control includes inhibition of c-Myc-dependent cellular transformation in vitro and loss of USF transcriptional activity in many cancer cell lines. However, a direct effect of USF on the tumorigenicity of an established cell line has never been demonstrated. Here, cell lines derived from rat embryo fibroblasts transformed by c-Ha-Ras and either c-Myc or E1A were used as model system to investigate the tumor suppression ability of USF. Overexpression of USF2 stimulated transcription and inhibited colony formation in c-Myc-transformed, but not E1A-transformed, fibroblasts. Stable clones expressing high USF2 levels were constructed from c-Myc-transformed fibroblasts. In two of these clones, overexpressed USF2 did not activate transcription, and there was no significant change in the transformed phenotype. In contrast, a clone that expressed transcriptionally active USF2 exhibited altered morphology and a strongly decreased ability to proliferate in semisolid medium. The ability of these cells to form tumors in nude mice was also decreased by a factor of more than 30 as compared to the parental cell line or cells overexpressing transcriptionally inactive USF2. Cotransfection assays with USF- or Myc-specific dominant-negative mutants indicated that active USF2 inhibited cellular transformation by preventing transcriptional repression by c-Myc.